Problems with the Specificity of the Primers - (Oct/28/2010 )

Hi All,

I designed TaqMan assays for various sequences. However I found that in some cases, my reverse primer was specific and did not bind to any other sequences on BLAST search and forward primer was entirely homologous. Will such a primer pair work? Also if 15/16 sequence bases are homlogous to other sequences, should I still consider it to be specific since 1 base should be enough to make my primer unique to amplify?

If you got one primer matched to many nonspecific sequences, then you maybe designed a primer in the repeat region. That's not very good, since the could be hundreds or even more of them and primer would anneal to all of them and all of them would elongate. If you're using primer3 to design primers, you should select Mispriming Library when you design them.

It depends on the position of the mismatch base, mismatch on the 5' end or in the middle doesn't matter at all, and usualy even 1 bp mismatch on the very 3' end isn't enough to stop nonspecific amplification as many alele-specific PCRs use 2 bp mismatch on the 3' to differ between two SNPs. So that primer is not specific.

It is hard to know without trying! Sometimes primer pair seem to be perfect design-wise but doesn't work in tube, sometimes primers seem to be nonsense due to various reasons but in tube the work perfectly. Luckily primers are not expensive so, I'd suggest to order a bunch of them and try them out. Then you can pick up the best ones and fine-tune it with optimizing cycling conditions and the right buffer.